1. Field of the Present Invention
The present invention relates to the collection of breath condensate medical testing and diagnosis, and, in particular, to a double-walled chamber having a coolant material embedded between the inner and outer walls, a side-mounted breath input assembly, an outlet from which condensate may be collected, and a plunger for expressing the condensate through the outlet. The collected sample may then be tested for biomarkers indicating the presence and severity of lung ischemia and associated pulmonary vasoconstriction.
2. Background
Approximately 6% of exhaled breath is water vapor and water droplets. One source of water in breath is from the fluids that line the alveoli of the lung. In other words, the water vapor exhaled from the breath equilibrates with fluid in the bronchi and alveoli, and therefore breath condensate collection provides a noninvasive means of sampling these fluids.
Exhaled breath condensate contains water soluble and water insoluble molecules, including dissolved gases, organic solutes, ions and proteins. Breath condensate samples from patients with certain diseases have been shown to contain elevated content of inflammatory molecules. For example, previous work has demonstrated that smoking, asthma and cystic fibrosis increase the presence of prostaglandin derivatives, thromboxane, leukotrienes and cytokines. (S A Kharitonov and P J Barnes, Exhaled markers of pulmonary disease, Am J Respir Crit Care Med 163:1693-1722, 2001.) Until recently, though, little work has been done to identify biomarkers in exhaled breath water vapor that may be able to assist in determining the presence and severity of lung ischemia.
However, recent research indicates that it may be possible to detect lung ischemia by performing a battery of tests on relatively small breath condensate samples. A proposed battery of tests for lung ischemia may include fibrinopeptides, thromboxane B2, platelet activating factor, leukotrienes C, D and E, carbon monoxide-to-nitric oxide ratio and chemokine and other proteins. Measurement of fibrinopeptides in breath condensate is believed to have the potential to allow more localized measurement of the presence of clot in the lung vasulature. It is publicly known that thrombin cleaves fibrinogen A peptide from fibrinogen as a prerequisite to fibrin gelation. Owing to its small size, it is hypothesized that fibrinopeptides will traverse the alveolar membrane, and equilibrate in alveolar fluid, and thus will be found in exhaled condensate.
It is also believed that pulmonary vascular constriction may be detected by measuring PGF2α, thromboxane B2, PAF, leukotrienes C, D, and E, and the ratio of CO to NO in condensate, thus providing a basis for initiating pulmonary vasodilator therapy or COX1,2 inhibition. Our laboratory has used an experimental pulmonary vascular occlusion (PVO), induced by venous infusion of polystyrene microspheres in a rat, to determine three major findings related to breath condensate analysis. We and others have found increased content of PGF2α, thromboxane B2, platelet activating factor (PAF) and vasoconstrictive leukotrienes C, D, and E in the lung washings in our rat model. (Nakos, Am J Resp Crit Care Med 1998, 158:1504) The magnitude of the concentration of these vasoconstrictive agents correlated with the severity of hypoxemia and pulmonary hypertension. We also have found extremely elevated expression of the gene encoding heme oxygenase-1 but the nearly complete absence of expression of the gene encoding for the inducible enzyme, nitric oxide synthase. Heme oxygenase produces carbon monoxide (CO) from heme substrate whereas nitric oxide synthase produces nitric oxide (NO). Both are vasodilator substances. In rats subjected to PVO, we have also found early increases in lung gene expression of cytokine induced neutrophil attractant 1 and 2 (CINC 1 & 2), and monocyte/macrophage chemoattractant protein (MCP) 1 and 2, and monocyte/macrophage inflammatory proteins (MIP) 1 α and 1 β with concomitant increases in each protein in the washings from the lung airways and alveoli obtained as soon as 2 hours after induction of PE, and lasting up to 18 hours after PE induction. The chemoattractant molecules can cause the migration of leukocytes into the affected area, and through this mechanism, can potentiate injury during therapeutic reperfusion.
Further, the presence of certain chemokines in exhaled condensate is believed to predispose reperfusion injury. The chemokines discovered in rats included CINC 1, CINC 2, MIP 1α, MIP 1β, and MCP 1 and 2. The human homologues that will be tested in our device will include CXCL1, CXCL 2 and CXCL 3; CCL 2, CCL 3, CCL 4 and CCL 8, using nomenclature outlined by Zlotnick and Yoshie, Immunity, 2000, 12:121-127. Chemokines have been found with an inflammatory model of pulmonary hypertension. (Kimura, Lab Invest 1998 78:571-81; Ikeda, Am J Physiol Heart Circ Physiol, 2002, 283(5):H2021-8). Unlike the in-vivo PVO model, which causes primarily obstructed blood flow, the model in the latter study incites inflammation and remodeling, which over weeks leads to vascular occlusion. Likewise, investigators have also found increased chemokine expression in lungs subjected to hilar ligation or clamping, which interrupts both perfusion and ventilation. The latter model differs significantly from in-vivo PVO because alveolar ventilation continues with in-vivo PVO. Thus the ischemic insult differs with in-vivo PVO versus hilar ligation.
Attempts have been made to analyze exhaled breath, including breath condensate, or otherwise measure certain components of exhaled breath. For example, U.S. Pat. Nos. 6,419,634 and 6,033,368 to Gaston IV et al. disclose a disposable device with a coolant coaxially surrounding a tube in order to cool exhaled breath sufficiently to cause condensation on the walls of the inner tube. Unfortunately, the device is designed for the measurement of nitrogen oxides and is not intended to facilitate protein or eicosinoid determinations on breath condensate. As a result, it suffers from a number of drawbacks. First, the Gaston device is mounted directly on the analyzer, and thus is too large and too cumbersome to use at the bedside for collection of small volumes of condensate in emergency department or other ambulatory patients. The Gaston device also suffers from inefficient sample collection inasmuch as the sample must be aggregated in one chamber and then transferred by the combined actions of droplet accretion and gravity to a separate second chamber for analysis. Perhaps worse, the Gaston device is incapable of use separate from the analyzer, in that the apparatus collects condensate in a chamber specifically designed for spectrophotometric analysis for nitrogen oxides, and thus has no port or other accessible reservoir from which condensate may be aspirated, aliquotted or otherwise withdrawn and subsequently transferred to a separate assay well to measure the components of the panel described above.
Further, although Gaston mentions the use of a device similar to a syringe plunger in expressing condensed fluid down its inner tube, Gaston fails to solve the problem of how to integrate such a plunger with the inlet tube. Also, even the inclusion of a plunger to express fluid down the inner tube of the Gaston device would still fail to solve the additional problem described previously; that is, Gaston still discloses no simple way to remove the fluid for removing and transferring the collected fluid for testing outside of the analyzing chamber. Finally, the Gaston coolant is not calibrated to permit condensation of a calibrated amount of condensate from a limited number of breaths. Instead, the Gaston device requires a lengthy period of sustained breathing in order to collect a sufficient quantity of condensate, a problem that is exacerbated by the absence of a plunger to more efficiently remove condensate from the device. This is due in large part to the considerable quantities of condensate that are necessary in Gaston for the intended type of testing to be performed thereon. As discussed previously, a major purpose of the present application is to collect relatively the small quantities of condensate necessary to perform the types of tests described above. Because such testing was not anticipated by Gaston, the Gaston device was not developed to permit such testing. Thus, a need exists for a fast and convenient apparatus and method for collecting small amounts of breath condensate in a manner that permits aliquotting as desired for the performance of tests such as the ones described above.